Fragile X syndrome (fraX), resulting from the loss of fragile X mental retardation protein (FMRP), is the most common cause of inheritable mental retardation. In addition to cognitive impairment, fraX is characterized by abnormal "stress-related" behaviors, and children with fraX have greater basal and stress-induced salivary levels of the adrenal hormone cortisol, as compared to unaffected siblings. These data suggest that hypothalamic-pituitary-adrenal (HPA) axis function is altered in fraX. A murine model of fraX has been developed that exhibits several features of this syndrome and holds promise for identifying the cellular and behavioral consequences of Fmr1 deletion. Work by the investigator has demonstrated that fragile X mental retardation 1 gene knockout (Fmr1-K0) mice also have greater responses to stress including elevated gene expression and glucocorticoid levels than do wild-type mice. These data indicate that Fmr1-KOs are exhibiting a hyper-stress response, similar to the phenotype in human fraX, yet more information is needed as to the extent the HPA axis is altered, the cellular basis of this dysfunction, and potential therapeutic targets. The goal of the proposed research is to obtain such information and lay the groundwork for understanding the contribution of an exaggerated stress response to cognitive impairment in fraX. Three aims are proposed. Specific Aim 1 will test the hypothesis that there is a generalized increase in HPA tone in fragile X mutants (Aims 1A & 1B), and that the disparity among genotypes is enhanced by chronic stress (Aim 1A). In Aim 1A, adrenocorticotropic hormone (ACTH) release and corticotropin-releasing factor receptor 1 (CRH-R1) mRNA levels will be analyzed at three ages (3 mo, 12 mo, 24 mo) in handled (unstressed) and stressed Fmr1-KO and WT mice. Aim 1B will examine basal, diurnal corticosterone fluctuations in Fmr1-KOs and WTs to determine if levels are altered as in fraX humans. Specific Aim 2 will test the hypothesis that immobilization stress will alter the subcellular compartmentalization of the glucocorticoid receptor in fraX mutants as compared to WT mice (Aim 2A) at the light microscopic level and, in particular, that there are greater levels of glucocorticoid receptor in nuclear fractions of cortical cells from Fmr1-KOs as compared to WTs under basal conditions and following stress (2B) using Western blot analysis. Specific Aim 3 will test the hypothesis that fraX mutant mice have an exaggerated stress-induced hyperthermic response, and that this is attenuated by antagonism of group I metabotropic glutamate receptor type 5 (mGluR5) function. The proposed research will build upon initial findings in the adult male Fmr1-KO to test the general hypothesis that in fraX there is a dysregulated HPA axis that leads to a heightened stress response, and that antagonism of the group I mGluR5 will reduce stress-related anxiety seen in this syndrome. Given that stress results in cognitive impairment and has been reported by some to increase dendritic spine densities, two attributes of fraX, studies on stress and the HPA axis in fraX may give valuable insight into the cause of mental retardation in this syndrome. [unreadable] [unreadable]